1. Field of the Invention
The present invention relates to an activation method of a protein derived from an extremely thermophilic bacterium in a nucleic acid amplification reaction, and a nucleic acid amplification method and a kit for amplifying nucleic acids using the same.
2. Description of the Related Art
A polymerase chain reaction (Hereinafter, simply referred to as “PCR”) is a remarkable technique which makes it possible to amplify a target specific DNA region 100,000 times or more in a short time. However, it is difficult to optimize the reaction. That is, the problem is that non-specific amplification occurs due to mis-priming such as annealing of the primer to sites other than the target sequence or annealing between primers. Therefore, it is needed to establish a technique which makes it possible to amplify the target DNA specifically. To achieve this, it is needed to control PCR so that no mis-priming occurs at each step of the PCR cycles.
In this regard, the hot start method is known as a method for making it difficult to cause non-specific amplification of nucleotide 5′-triphosphate. According to this method, a procedure is used such that a thermostable DNA polymerase does not undergo an extension reaction until the temperature reaches the annealing temperature or more. Specifically, the procedure is carried out by a method in which an antibody is bound to a DNA polymerase to inhibit the activity, or the like.
Also, recently, the present inventors reported that a RecA protein derived from an extremely thermophilic bacterium (hereinafter, simply referred to as “RecA”) can interact with a template or a primer to promote binding of the primer only to a specific template sequence, thereby it can suppress mis-priming (for example, see WO 2004/027060). In the above-mentioned hot start method, the antibody protein used therein is denatured and deactivated in the first heating cycle of PCR. However, RecA suppresses mis-priming without losing its activity throughout the PCR cycles, which makes it possible to amplify only the specific PCR products.
Here, RecA is a protein which binds to a single-stranded nucleic acid cooperatively, searches a homologous region between the single-stranded nucleic acid and a double-stranded nucleic acid and undergoes homologous recombination of nucleic acids. Regarding RecA, various researches have been conducted for Escherichia coli (for example, see Watanabe R., et al., Interaction of Escherichia coli RecA protein with ATP and its analogues, J. Biochem., November 1994, Vol. 116, No. 5, pp. 960-966, and Ellouze C., et al., Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA-DNA complex by inactive nucleotides, Eur. J. Biochem., May 1999, Vol. 262, No. 1, pp. 88-94). For example, it has been reported that RecA requires ATP or dATP for its strand displacement activity, and the formation of a strong complex of RecA and DNA is necessary. Furthermore, it has been reported that adenosine 5′-O-3-thiotriphosphate has an influence on the strand displacement activity of RecA, and the contact between the chemical group at γ position of the nucleotide and the protein has an influence on the stability of a RecA-DNA complex.
However, these are all related to RecA derived from Escherichia coli, and there are almost no research reports on RecA derived from an extremely thermophilic bacterium which is used in the above-mentioned PCR technique reported by the present inventors. In addition, RecA derived from the extremely thermophilic bacterium and RecA derived from Escherichia coli are remarkably different in their properties, for example, resistance to a denaturant, thermostability, pH resistance, amino acid sequence and the like. Therefore, it was not possible to apply the technique for RecA derived from Escherichia coli as itself to RecA derived from the extremely thermophilic bacterium.
However, since in the PCR thermal denaturation is repeated under high temperature and the annealing temperature is high, even the thermostability of RecA derived from an extremely thermophilic bacterium is not sufficient. Thus, in the above-mentioned inventions that the present inventors reported previously, it was shown that the biological function possessed by RecA in the initial stage of PCR is reduced, and non-specific amplification occurs progressively through every cycle. At the same time, it was also shown that the biological function of RecA is reduced by a PCR reaction solution. As a result, mis-priming occurred and non-specific amplification was generated particularly at the later stages of the PCR cycles. Therefore, it was still required to establish a technique which makes it possible to control PCR further properly in each step of PCR.
In light of such circumstances, an object of the present invention is to provide a method which makes it possible to activate RecA and maintain its biological function in all the PCR cycles by improving the above-mentioned method. Furthermore, an object of the present invention is to provide a nucleic acid amplification method and a kit for amplifying nucleic acids, which makes it possible to suppress non-specific amplification more specifically and efficiently, and to amplify only a desired nucleic acid, by maintaining the biological function of RecA.